The present invention relates to an enzyme solution and a method for producing the same, an enzyme preparation obtained from the enzyme solution, a proteolytic enzyme preparation containing the enzyme solution or the enzyme preparation as an active component, and a strain producing the proteolytic enzyme.
In extremely diversified industrial fields, techniques of hydrolyzing various proteins into peptides or amino acids have been utilized widely. The techniques are applied to the preparation of, for example, medical enteric formulas and nutritious supplements as food materials. The techniques are also applied to increase the food utilization efficiency by enhancing the efficiency of hydrolyzing proteins hard to be hydrolyzed, which are contained in soybean protein and the like. Further, the techniques are utilized for the preparation of amino acid seasoning from proteins as raw materials. Still further, the techniques are utilized for the preparation of bread with enhanced rise.
Although chemical degradation methods using hydrochloric acid and the like so as to hydrolyze protein are efficient, the methods may result in the formation of undesirable by-products because of severe hydrolyzing conditions. Particularly in industrial fields of foods, seasonings, materials for nutrition and the like relating to human bodies, methods capable of hydrolyzing protein under mild conditions are prefered (for example, Japanese Patent Publication No. Hei-7-53106, Japanese Patent Laid-open No. Hei-11-75765, and the like).
Herein, proteolytic enzymes for industrial use for the purposes described above have been produced so far, mainly by means of various bacteria and fungi. However, it is generally said that proteolytic enzymes obtained from bacteria and those obtained from fungi have both advantages and disadvantages. Actually, proteolytic enzymes for industrial use having sufficient activity and stability have not been available.
In other words, bacteria-derived proteolytic enzymes generally have good heat resistance but have low peptidase activity for hydrolyzing protein into amino acids, or low potency of hydrolyzing proteins into low molecules. Therefore, the degradation products obtained by using bacteria-derived proteolytic enzymes contain great amounts of high-molecular peptides. Hence, such products have a disadvantage in that the bitterness thereof as food materials, seasonings and the like is strong, and in that the intestinal absorption thereof as materials for nutrition is slow. Although bacteria-derived proteolytic enzymes have good heat resistance, there has hardly been reported such proteolytic enzyme with high heat resistance that can retain peptidase activity, for example, in a high temperature range of about 60xc2x0 C.
In contrast, fungi-derived proteolytic enzymes are generally excellent in peptidase activity, wide cleavage specificity of peptide bond, potency of hydrolyzing proteins into low molecules and the like. However, the heat resistance, for example, the potency of protein hydrolysis in a moderate to high temperature range of about 50xc2x0 C. or higher, is poor. Therefore, such proteolytic enzymes essentially require protein hydrolyzing steps in a relatively low temperature range, readily allowing the propagation of contaminated microorganisms.
As described above, there have not been provided proteolytic enzymes with high thermal stability such that the enzyme is never inactivated in moderate to high temperature ranges and also with great potency of hydrolyzing proteins into low molecules.
Further, it is desired to effectively hydrolyze proteins hard to be hydrolyzed, such as those contained in soybeans and the like, as useful protein materials. It is thus important that proteolytic enzymes have wide cleavage specificity for peptide bonds in protein. However, no proteolytic enzyme has been provided, which has both the thermal stability and the great potency of hydrolyzing proteins into low molecules and which additionally has high potency of hydrolyzing proteins hard to be hydrolyzed.
The present inventors cultivated a bacterium of genus Bacillus, which was isolated from the dough of xe2x80x9cMantou (a kind of Asian steamed buns)xe2x80x9d as one of traditional foods in the Mongol District. It was then found that the enzyme solution consequently obtained had the enzyme characteristics as described above.
Based on the findings, the invention provides a proteolytic enzyme solution with thermal stability in moderate to high temperature ranges and with excellent potency of hydrolyzing proteins into low molecules as well. Further, the invention provides a proteolytic enzyme solution additionally having the potency of effectively hydrolyzing protein hard to be hydrolyzed. Still further, the invention provides a method for producing such enzyme solution. Further, the invention provides an enzyme preparation obtained by separating the enzyme protein from the enzyme solution. And yet further, the invention provides a proteolytic enzyme preparation containing the enzyme preparation as the active component, which can be used for given uses. Still further, the invention provides a strain producing proteolytic enzyme to prepare the enzyme solution and the enzyme preparation.
The enzyme solution of the invention is an enzyme solution with proteolytic activity, which can be obtained by cultivating a bacterium of the genus Bacillus, and has such highly heat-resistant peptidase activity that the residual activity of the enzyme after 1-hour heat treatment at 60 to 65xc2x0 C. at pH 7 is substantially 100%. The use of the enzyme solution enables the protein hydrolyzing process under temperature conditions never permitting the propagation of contaminated microorganisms, for example at 50xc2x0 C. or higher, or in moderate to high temperature ranges up to 60 to 65xc2x0 C. Further, the peptidase activity can allow sufficient potency of hydrolyzing proteins into low molecules. The peptidase activity is more preferably an aminopeptidase activity.
More preferably, the enzyme solution of the invention additionally has protease activity and collagenase activity in combination. The protease activity includes neutral protease activity with favorable pH within the neutral region and alkaline protease activity with favorable pH within the alkaline region. Such enzyme solution can be expected of more excellent potency of hydrolyzing proteins into low molecules and the large initial reaction velocity as a general characteristic of protease activity. Therefore, proteolytic reaction can be speedily promoted. For meat tenderization, for example, meat tenderization by the peptidase activity and the protease activity can progress, simultaneously with the cleavage of collagen in the connective tissues by the collagenase activity, so that very tasty meat can be prepared.
Still more preferably, the enzyme solution of the invention has at least one of the following characteristics 1) to 4) so as to provide additional effects described below.
1) Enzyme solution with peptide cleavage site specificity capable of cleaving sites of at least ten amino acid types bonded in the peptide chain of protein. Regarding such wide range of peptide cleavage site specificity, peptide cleavage site specificity capable of cleaving 10 or more, preferably 12 amino acid types bonded at the carboxyl termini can be observed. Such amino acid types specifically include leucine, isoleucine, phenylalanine, lysine, valine, alanine, threonine, glycine, serine, glutamine, asparagine and arginine.
Typically, general proteolytic enzymes have peptide cleavage site specificity capable of substantially cleaving 5 to 6 or less, at most less than 10 amino acid types bonded. Therefore, the peptide cleavage site specificity of the enzyme solution covers a very wide range. Thus, the enzyme solution can retain great potency of hydrolyzing proteins into low molecules. Probably owing to the wide range of peptide cleavage site specificity, the enzyme solution has effective proteolytic activity over proteins, such as those derived from for example soybeans, which have been so far hard to be hydrolyzed.
2) Enzyme solution with such potency of hydrolyzing proteins into low molecules that the 17-hr hydrolysis by use of the enzyme solution of 200 units on a protease activity basis per 1 g of acid casein generates peptides or amino acids with molecular weights of 1,000 or below in an amount of 50% by weight or more relative to the acid casein. Regarding the potency of hydrolyzing proteins into low molecules, it has been found that the amounts of generated peptides or amino acids with molecular weights not exceeding 1,000 are prominently large, compared with conventional proteolytic enzymes.
Owing to such characteristics of the enzyme solution, the enzyme solution can efficiently generate peptides or amino acids with molecular weights not exceeding 1,000 from raw protein materials. Hence, high-quality amino acid materials and amino acid seasonings without bitterness can be prepared. Further, materials for nutrition with great absorbency from the digestive tract can be prepared.
3) Enzyme solution with the potency of hydrolyzing proteins into low molecules as described above in 2), that is exerted equally under temperature conditions of 45xc2x0 C. close to the relatively moderate temperature range and 60xc2x0 C. in a fairly high temperature range. Owing to such characteristics of the enzyme solution, the process of hydrolyzing proteins into low molecules can be progressed under temperature conditions never permitting the propagation of contaminated microorganism.
4) Enzyme solution with a 50% or more of solubilization ratio of soybean protein hard to be hydrolyzed under given conditions. It is expected that this proteolytic activity to hydrolyze protein hard to be hydrolyzed is also effective for hydrolyzing proteins in other species, such as meat connective tissue. This characteristic may possibly be related to the wide range of peptide cleavage site specificity, particularly cleavage site specificity to bonded sites of amino acid, such as glycine, valine and asparagine, which has not been found in conventional proteolytic enzymes.
Owing to such characteristic property, the enzyme solution can effectively hydrolyze soybean useful as a raw protein material, including its protein hard to be hydrolyzed, into amino acids.
The enzyme solution of the invention can readily and securely be obtained by cultivating a given bacterium of the genus Bacillus. The enzyme solution can be obtained by cultivating for example Bacillus subtilis M2-4 strain internationally deposited as FERM BP-7155 under the Budapest Treaty. The cultivation conditions therefor are not specifically limited. The strain may satisfactorily be cultivated by using general nutritious culture media under general conditions, and may also be cultivated in a specific culture medium under specific cultivation conditions, if necessary.
As the enzyme solution, a liquid culture medium where the strain is cultivated can be used without removing bacteria. Otherwise, an enzyme solution prepared by removing the bacteria or solids therefrom by means of filtration or centrifugation can also be used. The enzyme solution may further be concentrated by mild means using an ultrafiltration membrane.
From the enzyme solutions described above in accordance with the invention are obtained plural types of protein fractions with different molecular weights by column chromatography. By individually separating the proteins in these individual fractions and subjecting the proteins to enzyme activity test, it was confirmed that the enzyme solution contained aminopeptidase, neutral protease, acid protease and collagenase. Hence, the highly heat-resistant peptidase activity, protease activity, collagenase activity and the characteristics 1) to 4) described above are essentially based on the actions of these enzymes. However, how and to what extent each of the enzymes is involved in the individual characteristics 1) to 4) described above have not yet been identified accurately.
The invention further provides a method for producing an enzyme solution, comprising cultivating a bacterium of the genus Bacillus, and obtaining the enzyme solution of the invention from the culture. The method for producing an enzyme solution enables ready and secure obtainment of the enzyme solution.
Further, the invention provides an enzyme preparation obtained by separating the enzyme protein from the enzyme solution. For the separation of the enzyme protein, the enzyme protein can be separated by known appropriate processes, such as salting out of the enzyme solution under the saturation of ammonium sulfate and ethanol precipitation. The enzyme preparation may be obtained as crude enzyme powder or formulations of buffer solutions thereof. The enzyme preparation is advantageous in that the high-quality preparation is made more easily, compared with the enzyme solution. These enzyme preparations have the same enzyme activity and characteristics as those of the enzyme solution.
Still further, the invention provides a proteolytic enzyme preparation containing the enzyme solution or the enzyme preparation as the active component, which can be used for any of uses, such as hydrolysis of protein hard to be hydrolyzed, production of amino acid seasoning, bread production, meat tenderization, peptide production, production of protein with reduced allergenicity and cheese production.
The proteolytic enzyme preparation has highly heat-resistant peptidase activity, excellent potency of hydrolyzing proteins into low molecules, collagenase activity and the like in the above-mentioned various uses, which have not been found in the conventional proteolytic enzyme preparations. For use in bread production, the proteolytic enzyme preparation brings about an effect of volume increase (enhancement of rising).
Furthermore, the invention provides a strain producing proteolytic enzyme. The proteolytic-enzyme-producing strain provides effective means for producing the variety of the enzyme solution, the enzyme preparation and the proteolytic enzyme preparation. The proteolytic-enzyme-producing strain belongs to the genus Bacillus and is preferably a bacterium of the species subtilis. The most typical proteolytic-enzyme-producing strain is Bacillus subtilis M2-4 strain.
The Bacillus subtilis M2-4 strain was deposited as FERM P-17388 at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, the Ministry of International Trade and Industry (Address: 1-1-3, Higashi, Tsukuba, Ibaraki, Japan) on May 12, 1999. Then, the strain was transferred to the International Deposit as FERM BP-7155 on May 11, 2000 under the Budapest Treaty.
The Bacillus subtilis M2-4 strain is practically very useful, in particular, among plural species of strains producing proteolytic enzyme, which are isolated from the dough of Mantou, a traditional naturally fermented wheat food in the Mongol District. As a result of the identification test, it was found that the M2-4 strain exhibited the growth state in Table 1 (colony cultivated in the general agar culture medium manufactured by Eiken Chemical Co., Ltd. at 30xc2x0 C. for 48 hours) The bacteriological morphology in Table 2 was also observed. Further, it was found that the strain exerted the physiological properties in Table 3.
Based on the above-mentioned grounds, the bacterial strain was identified to belong to xe2x80x9cBacillus subtilisxe2x80x9d and was designated xe2x80x9cBacillus subtilis M2-4 strainxe2x80x9d.